The present invention relates to a novel chemokine-like protein of type CC, a DNA encoding said protein, a vector comprising said DNA, and a transformant containing said vector. In addition, the present invention relates to a use of the above novel protein, as well as a pharmaceutical composition comprising the protein, for example, an anti-inflammatory agent, an immune response modifier, or a diagnostic for diseases associated with inflammation and/or immunity. The present invention also relates to a monoclonal antibody raised against said protein and a hybridoma capable of producing said antibody.
Various exogenous or endogenous tissue impairments, invasions, or exposures to antigens, which are caused by physical, chemical or biological mechanisms, induce strong inflammatory or immune responses. Although these responses are essential biophylactic reactions, they may sometimes bring about acute or chronic diseases. When a substance causative of inflammatory or immune responses attacks a tissue, inflammatory or immune competent cells such as neutrophils, granulocytes, or macrophages, first adsorb to vascular endothelium, and then migrate outside the blood vessel and accumulate at an invaded or impaired tissue, or a tissue where an antigen exists. As substances inducing such sequential cell migration reactions, there exist a group of chemotactic cytokines, so-called xe2x80x9cchemokinesxe2x80x9d. Chemokines belong to a cytokine family which induces the migration reactions (chemotactic reactions), and at least 18 kinds of chemokines have been reported in human. Chemokines are known to be closely related to each other in the structure because they have similar amino acid sequences.
Chemokines are generally classified into two groups based on the arrangement of the first two cysteine residues of the four cysteines commonly conserved, that is, type xcex1 or CXC in which the two cysteines are intervened by one amino acid, and type xcex2 or CC in which the two cysteines are contiguous. Examples of the type CXC chemokines in human include IL-8, xcex2-TG, PF-4, MGSA/GRO, ENA-78, NAP-2, GCP-2 and IP-10, which mainly induce the activation and migration of neutrophils. Examples of the type CC chemokines in human include MIP-1xcex1, MIP-1xcex2, RANTES, MCP-1, MCP-2, MCP-3, and I-309, which mainly induce the activation and migration of monocyte/macrophages. Furthermore, the type CC chemokines also include those capable of inducing the activation and migration of T-cells, basophils, eosinophils, or the like (J. J. Oppenheim et al., Annu. Rev. Immunol. 9: 617-648, 1991; M. Baggiolini and C. A. Dahinden, Immunol. Today 15: 127-133, 1994). In addition, a chemokine SCM-1, which is presumed to be of type xcex3 or C different from the above-mentioned two types, has recently been reported (T. Yoshida et al., FEBS Letters 360: 155-159, 1995).
Since chemokines are profoundly involved in bioprophylactic reactions as described above, the identification of a novel chemokine and the elucidation of its activity would highly contribute not only to the analysis of the immune response in which the chemokine is involved, but also to the development of a method for treating, preventing, or diagnosing the related diseases. A chemokine derived from human is useful for such purpose though, those from animals other than human, preferably mouse, are also needed for conducting requisite tests using experimental animals. It is therefore desirable to obtain a novel chemokine of human origin and its mouse counterpart. As is often the case with many physiologically active substances exhibiting the activity in only a slight amount, a novel chemokine cannot be obtained in ease.
Considering the fact that chemokines are secretory proteins which share the similarity of structure, the present inventors have concentrated their efforts on obtaining a novel chemokine by means of a signal sequence trap method.
Thus, by using a signal sequence trap vector originally constructed (Yoshida et al., FEBS Letters 360:155-159,1995), a great number of cDNA fragments encoding secretory proteins or type I membrane proteins were separated from mitogen-stimulated normal human peripheral blood mononuclear cells, and their base sequences were compared with existing databases to find a cDNA fragment which potentially encodes a sequence characteristic of type CC chemokines.
A full-length cDNA was then isolated using the cDNA fragment, sequenced and identified. Finally, the inventors have obtained a DNA encoding a protein having a signal sequence which, after cleavage of the signal sequence, affords a secretory protein belonging to the mature type CC chemokines. The novel DNA was inserted into an appropriate expression vector, which was then transformed into an appropriate host cell. The transformants, when cultured, produced a type CC human chemokine-like protein capable of inducing the cell-migration of HUT78, and the like. The present inventors have succeeded in the isolation of mouse genomic DNA and cDNA using the DNA encoding human-derived chemokine-like protein, and, eventually, in the expression thereof in transformants. A base sequence of a DNA encoding a novel type CC chemokine-like protein from human and an amino acid sequence deduced therefrom are shown in SEQ ID NOS: 1 and 2. A base sequence of a DNA encoding a novel type CC chemokine-like protein from mouse and an amino acid sequence deduced therefrom are shown in SEQ ID NOS: 3 and 4.
The present invention provides a protein having the following characteristics:
1) its expression from peripheral blood mononuclear cells is induced in the presence of an immunological stimulus;
2) it is expressed mainly from thymus not from spleen, in the absence of the stimuli; and
3) it has the two contiguous cysteine residues characteristic of type CC chemokines.
The novel type CC chemokine-like protein of the present invention is expressed from peripheral blood mononuclear cells in the presence of an immunological stimulus, and the expression can be accelerated by phytohemagglutinin (PHA) or under the condition where the humoral immunity is induced. Particularly, the expression from monocytes is induced to higher extent in the presence of a cytokine selected from GM-CSF, IL-3 and IL-4 which participate in the induction of humoral immunity. The protein, as a characteristic, is not induced by TNF-xcex1 or IFN-xcex3 which induces the cellular immunity, or LPS.
Based on the characteristics of the novel protein of the present invention that the organ which constitutively expresses it is thymus and that the production thereof is induced in response to an immunological stimulus, the present inventors named the protein xe2x80x9cTARCxe2x80x9d (Thymus and Activation-Regulated Chemokine). Accordingly, the novel protein and the DNA encoding it are referred herein to as xe2x80x9cTARCxe2x80x9d or xe2x80x9cTARC proteinxe2x80x9d, and xe2x80x9cTARC DNAxe2x80x9d, respectively. The term xe2x80x9cTARCxe2x80x9d includes those derived from human and mouse; and if necessary, TARC of human origin may be referred as xe2x80x9chuman TARCxe2x80x9d or xe2x80x9chTARCxe2x80x9d, and that of mouse origin as xe2x80x9cmouse TARCxe2x80x9d or xe2x80x9cmTARCxe2x80x9d. Similarly, DNAs encoding these proteins are referred as human TARC DNA, hTARC DNA, mouse TARC DNA, or mTARC DNA, respectively. However, human TARC and the DNA encoding it may sometimes be referred simply as TARC and TARC DNA, respectively. In addition, in the present specification, the terms xe2x80x9cDNAxe2x80x9d and xe2x80x9cgenexe2x80x9d encoding TARC are used interchangeably. The DNA may be a synthetic or natural DNA.
Once a DNA encoding TARC is disclosed by the present invention, those one ordinary skilled in the art can easily obtain a TARC variant with a function and/or an activity substantially equivalent to that of the TARC having the above amino acid sequence, through the substitution, insertion or deletion of one or more amino acids by a method known in the art. A variant so obtained is, therefore, also included within the scope of TARC of the present invention.
Accordingly, the present invention also provides a human type CC chemokine-like protein comprising an amino acid sequence of the amino acid residues 24-94 in SEQ ID NOS: 1 and 2, and a variant thereof which containes, in said sequence, a substitution, insertion or deletion of an amino acid(s) or an amino acid sequence(s) and which has a function or an activity substantially equivalent to that of the said human type CC chemokine-like protein.
The present invention also provides a human type CC chemokine-like protein comprising an amino acid sequence of the amino acid residues 1-94 in SEQ ID NOS: 1 and 2, and a variant thereof which contains, in said sequence, a substitution, insertion or deletion of an amino acid(s) or an amino acid sequence(s) and which has a function or an activity substantially equivalent to that of the said human type CC chemokine-like protein.
Furthermore, the present invention also provides a mouse type CC chemokine-like protein comprising an amino acid sequence of the amino acid residues 24-93 in SEQ ID NOS: 3 and 4, and a variant thereof which contains, in said sequence, a substitution, insertion or deletion of an amino acid(s) or an amino acid sequence(s) and which has a function or an activity substantially equivalent to that of the said mouse type CC chemokine-like protein.
In addition, the present invention also provides a mouse type CC chemokine-like protein comprising an amino acid sequenced of the amino acid residues 1-93 in SEQ ID NO: 2, and a variant thereof which contains, in that sequence, a substitution, insertion or deletion of an amino acid(s) or an amino acid sequence(s) and which has a function or an activity substantially equivalent to that of the said mouse type CC chemokine-like protein.
A xe2x80x9cvariantxe2x80x9d of TARC described in the present specification may contain chemical or biological alterations, or natural or artificial amino acids, so far as it exhibit a function or an activity substantially equivalent to that of TARC having the amino acid sequence described herein.